The Preliminary Study For Overcoming The Delayed Xenograft Rejection By Using RNAi To Specially Suppress The Expression Of NF-κB P65 In Mice

Background:Allotransplantation is nowadays a generally accepted treatment for organ failure, and xenotransplantation, i.e. transplantation of cells, tissues or organs between individuals of different species, is considered a promising, possible solution to the chronic lack of donor organs. Organs of phylogenetically-distant species are subject to much more severe and irreversible rejection reactions than that in allograft. Xenotransplantation rejection includes four stages: hyperacute rejection (HAR), delayed xenograft rejection (DXR), acute cellular rejection (ACR) and chronic graft dysfunction (CGD). It is generally acknowledged that there are methods to overcome the first major barrier of xenotransplantation (HAR), for example, the availability of transgenic pigs and antibody inhibition. The second immune barrier is the delayed or acute vascular xenograft rejection (DXR or AVR) in which the activation of endothelialⅡcells is the key factor of xenograft rejection and graft dysfunction, and the NF-κB takes an important role in the activation of endothelialⅡcells. NF-κB is often associated with immune reaction, thymus gland growth, embryogenesis, inflammation, acute reaction, cells reproduction, apoptosis, viral infection and other diverse pathological processes. Though suppression of the activation of endothelialⅡcells by inhibiting the expression of NF-κB were reported in other fields, there are few studies about NF-κB in the research of heart xenotransplantation by now. Our objective is to delay or weaken delayed xenograft rejection by suppressing the expression of target gene NF-κB using in vivo siRNA in mice. Methods:1. siRNA directed against mouse NF-κB p65 was designed and chemically synthesized based on the oligonucleotide sequences from NCBI. For image identification purpose, a part of the siRNAs were carboxyfluorescein (FAM)-labeled.2. EOMA cell was divided into three groups: (1) blank control group, (2) negative control group, (3) NF-κB siRNA group. One day before transfection, EOMA cell was reseeded in plate. 24h-72h after transfection, total RNA was isolated from EOMA cells using TRIzol Reagent, the expression level of NF-κB p65 mRNA was evaluated by RT-PCR. Total protein was isolated from EOMA cells, and the expression level of NF-κB p65 protein was evaluated by Western blot.3. To determine the tissue distribution of siRNA in mice, FAM-labeling siRNA/in vivo-jetPEI complexes (included 2 OD siRNA-NC) were injected into mice through jugular vein or tail vein, and the epifluorescence activity was examined in various organs 48h later.4. The mice were, via jugular vein, treated with single dose i.v. injections of NF-κB p65 siRNA/in vivo-jetPEI complex (dose: 1OD). Four different organs were dissected at five time points (from 1h to 1w) for the examinations by epifluorescence microscopy. A single dose of NF-κB p65 siRNA/in vivo-jetPEI complex (dose: 2OD) was injected into immune competent mice, then we dissected heart tissue at four time points (from 1d, 3d, 5d, to 7d) for the examination of NF-κBp65 mRNA by RT-PCR and compared the levels of NF-κBp65 mRNA at different time points.5. Correlate the siRNA uptake and distribution with the efficacy of RNAi in particular organs in the following experiments. The mice (3 per cohort) were, via jugular vein, treated with a single dose i.v. injections of NF-κB p65 siRNA/in vivo-jetPEI complex (dose: 1OD, 2OD, 3OD and 4OD siRNA and the volumes of in vivo-jetPEI according with the ratio of N/P =6). An additional group of mice (the sham operated group) were treated in parallel with PBS solution for unspecific effects. RT-PCR was applied to demonstrate the RNAi silencing in the heart after 72h. 6. The mice in the research were divided into 4 groups. Three animals in each group were injected with NF-κB p65 siRNA-/in vivo-jetPEI complex (dose: 2OD). The conditions of the survival, appetite and vigor of each animal had been observed for 7 consecutive days, and a comprehensive clinical biochemistry test was performed 1, 3, 5 and 7days after injection.7. The mice were, via jugular vein, treated with single dose i.v. injections of siRNA/in vivo-jetPEI complex (included 2OD NF-κB p65 siRNA or siRNA-NC). RT-PCR was applied to demonstrate the RNAi silencing in the heart after 24h. The levels of NF-κBp65, ICAM-1, VCAM-1 and IL-1a mRNA were evaluated.8. The mice were treated with siRNA/in vivo-jetPEI complex (included 2OD NF-κB p65 siRNA or siRNA-NC) via jugular vein. 48h after mouse-to-rat cardiac heterotopic xenotransplantation in neck, survival time, pathological changes, and levels of NF-κBp65, ICAM-1, VCAM-1 and IL-1a mRNA in xenografts were investigated. Serum IgM/IgG concentration in rat before operation were compared with those after operation.Results:1. NF-κB p65 siRNA and siRNA-NC were as capable as absorbed in EOMA cells under the help of the vehicle lipofectamine2000, and the transfection efficiency is nearly 90%. Distinct from siRNA-NC, after the NF-κB p65 siRNA transfection, the silencing of gene expression in EOMA cells could be observed.2. Luc activity of siRNA in the liver and kidney was much higher than that in the heart and lung 48 hours after tail vein injection. In animals with jugular vein injection the Luc activity was high in the heart and lung, similar to those in the liver and kidney.3. A single dose of FAM fluorescently labeled siRNA-NC combined with in vivo-jetPEI (included 2OD siRNA-NC) was injected into ICR mice, and four different organs were dissected at five time points (from 1h to 1w) for the examinations by epifluorescence microscopy. An initial microscopic analysis revealed that fluorescence was detectable only in the lungs and livers 60 min post-treatment with siRNA-NC/PEI complex. The FAM-fluorescence appeared in all tissues 24h after complex treatment, though the fluorescence was not strong. The intensity of FAM-fluorescence of the four organs (heart, lung, liver and kidney) increased to the highest point between 48h and 72h after injection,and decreased again 1w after injection.4. The mRNA levels in each group after siRNA treatment demonstrated that the NF-κB p65 mRNA levels decreased significantly (>50%) in 7 days (from 1d, 3d, 5d, to 7d), which were statistically different from the sham operated group (P<0.05).5. Only in the samples derived from the animals treated with 2OD~4OD siRNA, the NF-κB p65 mRNA levels were significantly reduced (>50%, P<0.05), as revealed by the respective mRNA quantification. In these three groups the expression of the target gene were inhibited similarly (P>0.05), whereas in the 1OD group the expression of NF-κB were inhibited slightly (P>0.05).6. Observed for 7 consecutive days after injection, the conditions of the survival, appetite and vigor of each animal had not been impaired till the observation time point. All the biochemical parameters evaluated were in the normal range as compared with those of the normal animals except the ALT value on day 1. There was a transient increase up to 337 of the ALT value on day 1 in the animals injected with complex, and the ALT value falls to a normal range 3 days after the injection.7. The levels of NF-κB p65, ICAM-1, VCAM-1 and IL-1a mRNA decreased markedly after the mice were treated with 2OD NF-κB p65 siRNA.8. The MST of xenograft in group without treatment was (1.80±0.83) d, that in control group was (1.60±0.87) d (vs. group without treatment, P>0.05). The MST of xenograft in experiment group was (5.17±1.63) d (vs. group without treatment, P<0.01).9. Cardiac muscle cells of xenograft before rejection in negative control group and experiment group lined up in order, light edema were seen in some cells, inflammatory cell and lymphocyte infiltration was detected. Endothelium of graft in negative control group exhibited typical swelling cytoplasm, but apoptosis in experiment group by electron microscope. Chaos and severe edema of cardiac muscle cells were seen in xenograft in negative control group and experiment group after rejection. A lot of inflammatory cells and some lymphocytes infiltrated between cardiac muscle cells. Endothelium of graft both in negative control group and experiment group exhibited typical activated appearance (swelling cytoplasm, an expansion of the rough endoplasmic reticulum, indicating that the endothelium is metabolically active and not undergoing cell death by apoptosis) by electron microscope.10. With siRNA-NC transfection, NF-κB p65 mRNA level of graft increased lightly in control group before rejection, vs. normal cardiac muscle, p>0.05. After rejection NF-κB p65 mRNA level of graft increased markedly, vs. normal cardiac muscle, p<0.05. With NF-κB p65 siRNA transfection, NF-κB p65 mRNA level of graft decreased markedly before rejection, vs. normal cardiac muscle, p<0.05. After rejection NF-κB p65 mRNA level of graft was yet high, but vs. normal cardiac muscle, p>0.05.11. The VCAM-1 mRNA level of heart in negative control group increased after transplantation (siNCn VS N, p<0.05; siNCp VS N, p<0.01; siNCp VS siNCn, P<0.05). The VCAM-1 mRNA level of heart in experiment group decreased markedly after transplantation, and increased after rejection (siP65n VS N, p<0.01; siP65p VS N, p<0.01).12. The ICAM-1 mRNA level of heart in negative control group increased after transplantation (siNCn VS N, p<0.05; siNCp VS N, p<0.05; siNCp VS siNCn, P>0.05). The ICAM-1 mRNA level of heart in experiment group decreased markedly after transplantation, and increased after rejection (siP65n VS N, p<0.05; siP65p VS N, p>0.05).13. The IL-1a mRNA level of heart in negative control group increased after transplantation (siNCn VS N, p<0.01; siNCp VS N, p<0.01; siNCp VS siNCn, P>0.05). The IL-1a mRNA level of heart in experiment group decreased markedly after transplantation, and increased after rejection (siP65n VS N, p<0.05; siP65p VS N, p<0.01)14. Serum IgM,IgG concentration in rats had no deference between three groups whether pre- or post-transplantation(P>0.05), and increased markedly after transplantation than pre-transplantation(P<0.05) in each group.Conclusions:1. Cationic liposome Lipofectamine? 2000 could transfect chemically synthesized siRNA to EOMA cells with high effect. 2. Introduction of NF-κB p65 siRNA to EOMA cells could abrogate endogenenous gene expression, the siRNA designed in the experiment was functional in vitro and suitable for in vivo application.3. Jugular vein is an effective transfection route for gene delivery targeting for hearts.4. Administered with NF-κB p65 siRNA/in vivo-jetPEI complex by jugular vein injection leads to successful inhibition of expression of NF-κB p65. The minimum dose of siRNA was 2OD so that the NF-κB p65 mRNA levels decreased significantly (>50%).5. After 2OD NF-κB p65 siRNA administrated, the absorption of siRNA in heart of mouse increased to the highest point between 48h and 72h which was the best time for a mouse to be transplanted to a rat.6. It was safe to a mouse which was transfected a single dose of 2OD NF-κB p65 siRNA in vivo.7. Inhibition of protein transcription of NF-κB p65 by siRNA could efficiently suppress the expression of NF-κB's target genes.8. Being transfected with 2 OD NF-κB p65 siRNA extended the survival time of cardiac xenograft in mouse-to-rat model.9. Endothelium of graft in the siRNA treated mouse exhibited evidence of apoptosis before rejection and activation after rejection.10. Being administered with 2 OD NF-κB p65 siRNA led to inhibition of expression of NF-κB p65 and its target genes such as ICAM-1,VCAM-1 and IL-1αunder the internal environment after cardiac xenotransplantation.